Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications

Abstract

Triplex-forming oligonucleotides (TFOs) are short, single-stranded oligomers that hybridise to a specific sequence of duplex DNA. TFOs can block transcription and thereby inhibit protein production, making them highly appealing in the field of antigene therapeutics. In this work, a primer extension protocol was developed to enzymatically prepare chemical nuclease TFO hybrid constructs, with gene-silencing applications. Click chemistry was employed to generate novel artificial metallo-nuclease (AMN)-dNTPs, which were selectively incorporated into the TFO strand by a DNA polymerase. This purely enzymatic protocol was then extended to facilitate the construction of 5-methylcytosine (5mC) modified TFOs that displayed increased thermal stability. The utility of the enzymatically synthesised di-(2-picolyl)amine (DPA)-TFOs was assessed and compared to a specifically prepared solid-phase synthesis counterpart through gel electrophoresis, quantitative PCR, and Sanger sequencing, which revealed similar recognition and damage properties to target genes. The specificity was then enhanced through coordinated designer intercalators—DPQ and DPPZ—and high-precision DNA cleavage was achieved. To our knowledge, this is the first example of the enzymatic production of an AMN-TFO hybrid and is the largest base modification incorporated using this method. These results indicate how chemical nuclease-TFOs may overcome limitations associated with non-molecularly targeted metallodrugs and open new avenues for artificial gene-editing technology.

A copper(ii) peptide helicate selectively cleaves DNA replication foci in mammalian cells

“…Next, the cleavage mechanism for the DD-Cu II 2 LLD helicate was investigated using reactive oxygen species (ROS) scavengers. 59,60 Here, 3WJ was incubated with DD-Cu II 2 LLD, Na-Lascorbate, and a series of selective ROS scavengers (L-histidine, D-mannitol, L-methionine, and tiron (sodium catechol sulfate)), and the resulting cleavage proles were visualized by PAGE (Fig. 5).…”

“…This has previously involved the attachment of well-studied chemical nucleases to nucleic acid recognition moieties such as dsDNA groove binders, [40][41][42][43] non canonical DNA binders (specically triplex DNA 44 and G-quadruplexes 45 ) or sequence specic dsDNA targeting agents. [46][47][48][49] However, to date, no chemical nuclease, copper-derived or not, specically directed against 3WJs has been described.…”

Thiazole orange-carboplatin triplex-forming oligonucleotide (TFO) combination probes enhance targeted DNA crosslinking

“…1 However, limitations of their use include systemic toxicity and poor target discrimination. 2 In recent years, antigene technology 3 -specifically triplex-forming oligonucleotides (TFOs) [4][5][6] -has emerged as an ideal vector for directing traditional platinum agents. [7][8][9][10] These modern strategies target extended genomic tracts while overcoming shortcomings in the targeting limitations of small molecule platinum drugs.…”

Ferrocene as a potential electrochemical reporting surrogate of abasic sites in DNA

“…10 This method is particularly alluring since it is compatible with Darwinian evolution methods to identify functional nucleic acids and it is not limited in terms of size or nature of the functional groups that can be incorporated. [11][12][13][14][15][16][17][18][19][20] In the latter, modified oligonucleotides can be accessed either by primer extension (PEX) reactions or under PCR amplification conditions by substituting one or multiple canonical nucleotides with the corresponding unnatural counterparts. Natural or engineered polymerases are particularly tolerant to nucleotides bearing modifications attached at position C5 of pyrimidine or N7 of 7-deazapurine nucleotides or with simple backbone modifications but can also be coerced to incorporate nucleotides with sugar or unnatural base surrogates.…”